When tissues (normal and neoplastic) increase in size, they require the formation of microcapillaries (angiogenesis) to provide nourishment to sustain their growth. Constituents of these microcapillaries include most prominently endothelial cells, but also associated mesenchymal cells, fibroblasts, smooth muscle cells, and pericytes. Angiogenesis is important in normal processes, such as wound healing, but also in diseases such as cancer, psoriasis, diabetes, rheumatoid arthritis, and age-related macular degeneration.
There is a need for improved methods for studying microcapillaries in vitro in both normal and diseased tissues. A summary of presently known methods is provided in Staton, et al., “Current Methods for Assaying Angiogenesis in vitro and in vivo,” Int J Exp Path (2004) 85:233-248. In vivo models are useful but cumbersome. In vitro models are less cumbersome but also more artificial and less relevant.
In particular, there is a need for improved methods to predict the activity of anti-cancer drugs and other treatments which target the microvasculature of tumors. For example, bevacizumab (Avastin®) is an FDA-approved anti-cancer drug which targets the microvasculature of tumors. The wholesale cost of Avastin® is more than $40,000 for 10 months of treatment; yet only a relatively small percentage of patients derive substantial benefit. As stated by Ince, et al., “Association of k-ras, b-raf, and p53 Status with the Treatment Effect of Bevacizumab,” J Natl Cancer Inst (2005) 97:981-989, the identification of biomarkers that may predict which patients are most likely to respond to such treatment is of considerable interest.
The most commonly used in vitro methods involve isolating and culturing endothelial cells. Once the cells have been cultured, the effect of drugs (or other perturbations) may be studied, using a variety of cell proliferation and/or cell death endpoints. Examples of cell proliferation endpoints include radioactive thymidine incorporation, cell counting, BrdU incorporation, and colony formation. Examples of cell death endpoints include measurement of cellular ATP, mitochondrial reduction of MTT, metabolism and intracellular trapping of fluorescein diacetate (and loss thereof), loss of cell membrane integrity by dye exclusion, and more specific measurements of apoptosis, such as TUNEL assay or caspase expression. In some cases, previously-isolated endothelial cells have been co-cultured with previously-isolated other cells, and differential effects of drugs on the different cell populations have been studied.
Other in vitro methods are based on organ cultures. For example, see Staton, et al., supra). These include rat aortic ring, chick aortic arch, porcine carotid artery, placental vein disk, and fetal mouse bone explant.
Cell culture assays have clear disadvantages. First, they depend upon the isolation and culture of viable endothelial cells, which can be problematic particularly in the case of fresh human tumors. Once isolated and cultured, they are removed from the native microenvironment, in which factors released by the tumor cells (or normal cells, in the case of normal tissues) are not present. Although isolated tumor cells (or normal cells) could, in principle, be co-cultured, this would not approximate the spatial relation and cell-cell interactions existing in vivo. Existing organ cultures have similar limitations, in that, as stated by Staton, et al., supra, “the model in not truly representative of the microvascular environment encountered in tumor growth as the large number of different factors released by the tumor cells and the tumor cells themselves are not present.” See also Auerbach, et al., “Angiogenesis assays: problems pitfalls,” Cancer Metastasis Rev. (2000) 19:167-172.
Non-cell culture, non-organ culture, approaches to studying and predicting the effects of bevacizumab have been disclosed by Ince, et al., J Natl Cancer Inst (2005) supra. Ince attempted to correlate k-ras, b-raf, and p53 status with treatment effect of bevacizumab, but concluded that they “did not identify any subgroup of metastatic colorectal cancer patients who were more likely to respond to bevacizumab therapy.” In their discussion, Ince, et al., noted that “To date, few studies have assessed the potential utility of biomarkers in predicting which patients are more likely to respond to antiangiogenic therapy in the clinic” and that no markers had been yet found to be predictive of clinical benefit. These authors suggested that “biomarkers which summarize the effects of all angiogenic regulators may better predict patient outcome than the analysis of a single growth factor or signal induction pathway,” but did not suggest any in vitro methods for this purpose. Instead, they noted ongoing work in which patients themselves are used as experimental models for predicting their own outcomes.
In these studies bevacizumab (and/or other treatments) are administered to the patient on a trial basis and then “early” treatment effects are assessed by means of external diagnostic scanning (e.g., MRI) and/or post-treatment tumor biopsies, with histopathologic evaluation of treatment effects (e.g., Willett, et al., Nature Med (2004) 10:145-147. This approach has many obvious disadvantages, including expense of treatment, exposure of patient to potential toxicity of ultimately ineffective therapy, and the expense of diagnostic studies (e.g., MRI). Such studies also lack of ability to test multiple different treatments simultaneously without risk to the patient as is possible with in vitro methods.
Clearly, the lack of useful in vitro models in which to study human tumor microvasculature is an obstacle to the identification and development of newer, more effective treatment approaches targeting tumor microvasculature.